Membrane phospholipids control gating of the mechanosensitive potassium leak channel TREK1

Tandem pore domain (K2P) potassium channels modulate resting membrane potentials and shape cellular excitability. For the mechanosensitive subfamily of K2Ps, the composition of phospholipids within the bilayer strongly influences channel activity. To examine the molecular details of K2P lipid modulation, we solved cryo-EM structures of the TREK1 K2P channel bound to either the anionic lipid phosphatidic acid (PA) or the zwitterionic lipid phosphatidylethanolamine (PE). At the extracellular face of TREK1, a PA lipid inserts its hydrocarbon tail into a pocket behind the selectivity filter, causing a structural rearrangement that recapitulates mutations and pharmacology known to activate TREK1. At the cytoplasmic face, PA and PE lipids compete to modulate the conformation of the TREK1 TM4 gating helix. Our findings demonstrate two distinct pathways by which anionic lipids enhance TREK1 activity and provide a framework for a model that integrates lipid gating with the effects of other mechanosensitive K2P modulators.

MS spectra of lipid extraction from purified TREK1. Shown are positive (a,b) and negative (c,d) ion mode spectra with and without the indicated phospholipid standards (POPC intensity = 5.7e5, POPE intensity = 1.0e5, POPA intensity = 2.4e5). In the samples without standards, POPC, POPE, or POPA were not observed. Multiple peaks other than the standards were observed in the negative and positive ion mode spectra, but none corresponded to phospholipids (see Source Data). These likely represent contaminants in the protein sample. (e) GC-MS selected ion monitoring of 6 ions: m/z 329, 368, and 458 (masses unique to cholesterol TMS) and m/z 337, 363, and 468 contained in the spectrum of ergosterol-TMS derivative. The red trace represents the chromatogram of the lipid extract from the TREK1 sample. The chromatogram of the pure cholesterol standard (retention time of 16.69 min) is shown in blue and the chromatogram of the ergosterol standard (retention time of 17.59) in green. The red TREK1 extract trace shows the cholesterol internal standard but ergosterol is absent. The large peak at 21.7 minutes is a disaccharide, likely D-(+)-cellobiose. (f,g) A second replicate of the native MS experiment shown in figure 3c. (f) Native mass spectrum of TREK1 dimer with POPA (upper panel) and a corresponding deconvoluted spectrum (lower panel) showing multiple peaks corresponding to TREK1 dimer with up to 12 bound POPA molecules. (g) Under more activating conditions, the number of bound POPA lipids is reduced to four.

Characterization of TREK1 conformational movements.
A representation of the all-atom root mean square deviation (RMSD) between (a) the two asymmetric subunits, TM4a and TM4b, of the apo TREK1 cryo-EM structure, (b) the TREK1 apo TM4a subunit with a matched subunit from the TREK1 POPA structure, both in the TM4 "up" conformation, and (c) the TREK1 apo TM4b subunit with a matched subunit from the TREK1 POPE structure, both in the TM4 "down" conformation. (d,e) Aligned and overlaid representations of the selectivity filter structures from the TREK1 apo (purple), TREK1 POPA (yellow) and TREK1 POPE (orange) structures. Amino acid sequences for SF1 (d) and SF2 (e) are displayed. (f,g,h) Cryo-EM density and ion occupancy modeled within the selectivity filters for the (f) TREK1 POPA structure, (g) TREK1 apo structure, (h) and the TREK1 POPE structure.
Lipid induced movement of a key residue within the POPA upper lipid binding site. Cartoon representations of the upper lipid binding site from the TREK1 POPA structure, with the positions of the W275 sidechain from numerous other structures overlaid. (a) The W275 residue faces outward in structures of TREK1 in the TM4 "down" (TREK1 POPE, apo TREK1 TM4b) and TM4 "up" (apo TREK1 TM4a, PDB: 6CQ6) states. The lipid acyl chain in the TREK1 POPA structure occupies the outward facing position of the W275 sidechain and moves the W275 sidechain (navy) inward. (b,c) Cryo-EM density maps for the Apo TREK1 TM4a subunit (b) and a subunit from the TREK1 POPA structure (c), showing the change in position of the W275 residue (orange) in the presence of a bound POPA lipid (yellow). When reoriented inward, W275 occupies (d) the structurally identified binding site for the TREK1 activator ML335 (PDB: 6CQ8, green) or (e) the position of the isoleucine residue in a TRAAK gain of function mutant G126I (PDB: 4RUE, pink).
Pore blocking densities in the TREK1 structures. Structural models of TREK1, focused on the pore vestibule region for the (a) TREK1 POPE structure in the TM4 "down" state, due to the presence of a POPE lipid (orange) stretched out across both TM4 helices, (b) the apo TREK1 structure in the TM4 "asymmetric" state due to a DDM molecule blocking the upward movement of only one of the TM4, or (c) the TREK1 POPA structure in the TM4 "up" state with an open pore region. Note that the lipid densities in (c) are outside of the field of view of this figure and not shown. Magnified views of the cryo-EM densities for the modeled (d) POPE or (e) DDM molecules, derived from the C1 symmetrized cryo-EM maps after postprocessing in RELION. (f) A structural model of the less populated class in the TREK1 POPE dataset, with TM4 in the "up/down" state, showing the location of the unmodeled pore density located asymmetrically near the TM4 down helix. Magnified overlays of this density with either a POPE lipid (g) or a molecule of DDM detergent (h) are shown.
cryo-EM data processing scheme for the asymmetric TREK1 apo state. TREK1 in an asymmetric TM4 "up/down" state Supplementary Figure 7 cryo-EM data processing scheme for the TREK1 POPA structure. TREK1 in the TM4 "up" state.

Supplementary Figure 8
Cryo-EM data processing scheme for the TREK1 POPE structure. TREK1 in the TM4 "down" state (major group, 135k particles, left side) or TM4 "up/down" state (minor group, 59k particles, right side). * denotes the final C1 refinement, used to model the POPE lipid. ** denotes the final C2 refinement, used to model the TREK1 protein structure.